Methods commonly used for the separation of human blood cells include immunomagnetic beads, flow cytometry, blood cell separator and culture amplification. The immunomagnetic beads are coated with known antibodies, and then mixed with human blood. Antigen-positive cells adhere to the immunomagnetic beads, which are passed through a magnetic pipe. The magnetic beads are adsorbed on the wall of the magnetic pipe. After the cells not bound to the magnetic beads pass through, the magnetism of the pipe is removed, and cells bound to the magnetic beads are collected. This method is associated with relatively higher cost, and may not be available to low- and middle-income patients. It may also affect the activity of cells and the effectiveness of cell therapy.
The cell sorting function of methods using flow cytometry is accomplished by the cell sorter. The process consists of the following steps: a liquid column emitted from a nozzle is divided into a series of small water droplets; each is determined whether it will be sorted according to a parameter selected by the logic circuit; then the selected cell droplet is charged by a charging circuit; charged droplets carrying cells deflect when passing through an electrostatic field, and fall into the collector; the rest of the liquid is sucked out as waste. Certain types of instrument use a capture tube for sorting. There are cost and safety concerns for clinical uses of this method.
The method using blood cell separator is mainly used to separate peripheral blood cells. Patients are given a mobilization agent, then the patients' peripheral blood is cycled and filtered by a cell separator. Cells with a diameter within a certain range are collected for cell therapy. The mobilization agents used in this method, however, may increase treatment burden. The volume of separated fluid containing cells may also be too large for clinical use, and patients may bear certain life-threatening risks.
Culture amplification involves collecting human blood, adding reagents to the blood sample and culturing the sample in incubator. The cells are washed before use after about one week in culture. The prolonged time of tissue culture is often accompanied by high risks of contamination, in addition to unknown direction of stem cell differentiation.
Existing technologies of bone marrow and blood separation include those disclosed in Chinese Patent Application No. 200510130326.7, entitled “A method of separating cells and specialized cell separation medium.” Various adjustments are required to adapt the method for chicken, bovine, and human blood. There is also plenty of room for improving the accuracy of separating the desired cells. The use of surfactants and the unknown identity of isolated cells are additional concerns for applying this method in the clinic. Thus, the method so far has only been used for simple tests.
Another stem cell separation medium and method is disclosed in Chinese Patent Application No. 200610035900.5, entitled “A stem cell separation medium and its use for stem cell isolation.” To practice this method, it is necessary to prepare working solutions, and to adjust the density of cell suspensions because cells can only be collected within the density range of 1.083 g/ml. This method collects a heterogeneous population of cells, which may include few desired target cells for therapeutic purposes. For umbilical cord blood, this method may not effectively remove red blood cells, resulting in transplant rejection in patients.
Chinese Patent Application No. 200610114475.9, entitled “Kit for separation of bone marrow mononuclear cells” discloses yet another method. When used to directly isolate stem cells from blood, the kit does not generate enough cells for clinical use. Therefore, this method involves cell culture and risks high rate of contamination. Moreover, cell culture in vitro may not fully recapitulate cellular environment and cell expansion in vivo. Cells so produced may possess the stem cell or progenitor cell morphology but may not have the desired function for use in the clinic.
Chinese Patent Application No. 200610106875.5 discloses kits for in vivo separation of nucleated cells and their use. This kit uses HISTOPAGUE1077, a lymphocyte separation medium with a density of 1.077 g/ml. Most of the cells so separated are lymphocytes, and may contain only a minority of stem cells for therapeutic use. Clinical efficacy of this method is therefore diminished.
Chinese Patent Application No. 200710137781.9 discloses kits for bone marrow and umbilical cord blood stem cell isolation in vivo and uses of such kits. The kit contains lineage cocktail (Lin antibodies), resulting in increased cost and economic burden to low- and middle-income patients and their inability to afford cell therapy. In addition, Lin antibodies may result in a greater burden to wash isolated cells, and consequences of introducing Lin antibodies into the human body remain unknown.
The following methods for isolating stem cells have been used in clinic.
1. Sieving. For example, blood is directly passed through a blood cell separator which acts as a sieve to sort cells by size, and larger cells are selected for use in treatment. The selected cells include populations of erythrocytes, stem cells, leukocytes, lymphocytes, etc., and the resultant volume of cell suspension is approximately 50 ml-150 ml. It is not advisable to directly inject such a large volume of heterogeneous population of cells into the human body for treatment.
2. Labeling. The desired cells are labeled with antibodies for isolation. The labels are then washed off from isolated cells. The carcinogenicity of unknown labels remains to be determined.
3. Negative selection. Unwanted cells in the blood sample are labeled. This method does not require washing the label off the labeled cells, and therefore maintains the original activity of desired cells. Therefore, this method has its advantages and may achieve therapeutic effects. It is well known that cell populations in the blood are kept at equilibrium by complex mechanisms including negative feedback signaling. By negative selection, cells that are not desired for therapeutic use are removed, resulting in disturbances of cell population equilibrium and water/electrolyte imbalance. Additionally, cells are squeezed, diluted and washed in various solutions during centrifugation and separation, which may reduce cell activity and function. Therefore, it remains unknown whether stem cells so isolated are in the best conditions for use in treatment. Likely the cell number and survival rate are reduced, resulting in lower treatment efficacy, need of larger starting blood volume and increased pain in patients.
To improve existing methods for clinical use, the following aspects should be considered:
1. High treatment cost. Equipment used for cell separation (for example, blood cell separator and flow cytometer) is expensive, leading to high cost of treatment, and is not conducive to the promotion and popularization of the treatment options.
2. Low cell activity. Immunomagnetic beads and flow cytometry generate labeled cells. These sorting methods can only accommodate a narrow range of cells to be screened. Labeled cells may exhibit declined activity and viability. Moreover, the metabolism of the labels and consequences of such labels remaining in the human body are unknown.
3. Bulky cell suspension. Cell separator is often used to separate peripheral blood, and subjects are injected with mobilizing agents for 2-7 days before blood collection to mobilize stem cells from the bone marrow into the peripheral blood. The resultant cell suspension from a blood cell separator often exceeds 50 ml in volume, with considerable numbers of remaining erythrocytes. Such bulky, low quality cell suspension may only be used for autologous subcutaneous injection.
4. Long procedure for cell isolation. Cells are cultured for a week before use in the clinic. Such prolonged procedure may miss the best timing for treatment, compromise treatment effect, and risk high rate of contamination in culture.
5. Many traditional separation methods are complex and tedious, and demand high levels of training of professionals performing the tasks. There is a need for improvements in areas ranging from experimental methods to clinical applications. It may be difficult to scale up the traditional methods for large scale production. Traditional methods may also be associated with high experimental costs, high risks of contamination during operation, difficulty in storage and transportation of cell suspensions, limited quantity of isolated cell suspension, and inability to meet the high clinical demand.
Chinese Patent Application No. 200910187212.4, entitled “Human bone marrow, umbilical cord blood, peripheral blood cell processing kit and cell processing method,” discloses using 0.9% NaCl solution or PBS as a diluent, using 6% hydroxyethyl starch or 0.2-1% methyl cellulose as a precipitating reagent, and using a solution (density 1.075 g/ml) formulated with Ficoll and meglumine diatrizoate as a separation reagent. This application discloses a method for safe, effective and efficient separation of stem cells suitable for clinical use.